EP2585171B1 - Formation de jonctions neuromusculaires dans un système défini - Google Patents
Formation de jonctions neuromusculaires dans un système défini Download PDFInfo
- Publication number
- EP2585171B1 EP2585171B1 EP11772857.6A EP11772857A EP2585171B1 EP 2585171 B1 EP2585171 B1 EP 2585171B1 EP 11772857 A EP11772857 A EP 11772857A EP 2585171 B1 EP2585171 B1 EP 2585171B1
- Authority
- EP
- European Patent Office
- Prior art keywords
- human
- serum
- cells
- derived
- motoneurons
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5058—Neurological cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
- G01N33/48728—Investigating individual cells, e.g. by patch clamp, voltage clamp
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/105—Insulin-like growth factors [IGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/40—Regulators of development
- C12N2501/41—Hedgehog proteins; Cyclopamine (inhibitor)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/50—Cell markers; Cell surface determinants
- C12N2501/58—Adhesion molecules, e.g. ICAM, VCAM, CD18 (ligand), CD11 (ligand), CD49 (ligand)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/08—Coculture with; Conditioned medium produced by cells of the nervous system
- C12N2502/081—Coculture with; Conditioned medium produced by cells of the nervous system neurons
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
- C12N2502/1335—Skeletal muscle cells, myocytes, myoblasts, myotubes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/20—Small organic molecules
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2535/00—Supports or coatings for cell culture characterised by topography
- C12N2535/10—Patterned coating
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Definitions
- the invention relates to the field of cell culture, and, more particularly, to formation of neuromuscular junctions.
- NMJ Neuromuscular junction
- An in vitro (1) co-culture system composed of human motoneurons and skeletal muscle would be useful for studies ranging from understanding NMJ synaptogenesis, target generation for NMJ related diseases, screening therapeutic candidates and conducting drug toxicology evaluation.
- the advantages of human-based in vitro systems compared to in vivo systems reside in that they are much simpler and therefore easy to manipulate any factors, to dissect the mechanisms or pathways and to analyze the results.
- MN-muscle co-cultures have been described in Xenopus (1, 2), chick (3-5), mouse (6, 7) and rat (8, 9), as well as in cross-species investigations between mouse MN-chick muscle (7, 10), human stem cell-derived MNs-myotubes from C2C12 cells (11).
- MN-muscle co-culture systems use serum containing media and a biological substrate (3-5, 8, 9).
- NMJ formation has been (2) demonstrated between hESCs and C2C12 cells in a serum based system (11), as well as between hSCSCs and rat myotubes derived from embryonic skeletal muscles in a defined serum-free system (14).
- serum based system 11
- hSCSCs rat myotubes derived from embryonic skeletal muscles
- serum-free system 14
- no human based in vitro NMJ system, in which both MNs and myotubes were derived from stem cells presently exists. Accordingly, there is a need in the art for a human based system for NMJ formation that does not suffer from one or more of the above described drawbacks.
- the method comprises forming functional neuromuscular junctions between motoneurons and muscle cells by co-culturing one or more human motoneurons and one or more human muscle cells in a substantially serum-free medium as further defined in the claims.
- the method comprises suspending human skeletal muscle cells in a serum-free medium; suspending human motoneurons derived from human spinal cord stem cells in the serum-free medium; plating the suspended muscle cells and the suspended motoneurons onto an artificial carrier; and monitoring for formation of functional neuromuscular junctions.
- the embodiment is directed to synthetic mammalian neuromuscular junction comprising a human motoneuron functionally linked to a human muscle cell in a substantially serum-free medium.
- the human motoneuron can be functionally linked to the human muscle cell on an artificial surface.
- a preferred artificial surface has a silicon based monolayer substrate deposited thereon, which may, if desired, be deposited in a predetermined pattern.
- the substantially serum-free medium is completely serum free.
- Some examples of the substantially serum-free medium comprise at least one synaptogenesis promoting component and one or more trophic factors. NbActiv4 can be added to the serum-free medium.
- the medium comprises the components in Table 1.
- the human motoneuron cells are derived from human spinal cord stem cells and the human muscle cells are derived from human skeletal muscle stem cells.
- Some embodiments can include a synthetic substrate adapted to support at least one neuromuscular junction thereon.
- the synthetic substrate is preferably silicon based and more preferably is DETA.
- the synthetic substrate may be deposited on a support surface in a predetermined pattern if desired.
- the synthetic substrate may be coated on a carrier.
- an in vitro system for forming NMJs between human cells comprises an in vitro co-culture adapted to allow NMJs to form between human neurons and human muscle cells in a defined environment.
- the defined environment is preferably achieved by utilizing a co-culture medium in which the ingredients and quantities of those ingredients are known.
- the medium contains no serum.
- the co-culture can also be prepared on substrate that has a defined surface, such as by assembling a synthetic material onto an underlying surface for example. In some cases, the synthetic material can be assembled on the underlying surface according to a desired pattern.
- This disclosure reports the first human-based in vitro NMJ system which supports the differentiation of human stem cell derived motoneurons and SKMs and provides for functional NMJ formation.
- the system developed in this study by the co-culture of human stem cell-derived motoneurons and SKMs, provides a system closer to the human condition that is capable of addressing the above described drawbacks, as well as neurological and/or muscular disease modeling, drug discovery and regenerative medicine.
- the human neurons are MNs differentiated from human spinal cord stem cells and the muscle cells are human skeletal muscle stem cells.
- a suitable co-culture medium that can be used in the human NMJ formation system is comprised of the ingredients provided in Table 1.
- the scope of the invention is not limited only to these ingredients, nor is it required that every one of the ingredients be used in every embodiment. Ingredients may be added to or taken away from Table 1 without falling outside the scope of the invention.
- the combination of Neurobasal medium, B27, Glutamax, GDNF, BDNF, Shh, RA, IGF-1, cAMP, CNTF, NT-3, NT-4, Vitronectin and Laminin has been found to be able to support the growth, differentiation, and long-term survival of MNs derived from human stem cells (11, 14).
- Laminins are important components of the extracellular matrix that facilitates synaptogenesis (1). Specifically, ⁇ 2 laminins are concentrated at synaptic sites and are useful for their postnatal maturation (57). The addition of the G5 supplement to the co-culture medium has been found to significantly enhance myocyte proliferation. However, the continuous presence of these trophic factors, including BDNF, GDNF, NT-3, NT-4 and cAMP, was found to significantly down regulate agrin deposition along the neurites and at nerve-muscle contacts, thus preventing synaptogenesis (2).
- NbActiv4 media In a preferred preparation of NMJs, the trophic factors were gradually withdrawn and the culture was fed using only NbActiv4 media.
- the NbActiv4 media formula was generated by adding three ingredients, cholesterol, estrogen, and creatine to media containing Neurobasal, B27 and Glutamax (53). There is evidence that the addition of these ingredients can significantly promote synaptogenesis (53-56). Therefore, the co-culture was first plated in the co-culture medium to ensure the survival and growth of MNs and myocytes, followed by the gradual withdrawal of these factors which enabled the reciprocal induction between the MNs and myotubes that naturally occurs in development.
- the defined co-culture medium delineates the basis for the essential components during NMJ formation, and provides a basic system for dissecting the individual factors, for investigating the underlying mechanisms, and later for treatment of diseases related to the cellular components of NMJs.
- a preferred substrate is trimethoxysilylpropyldiethylenetri-amine ("DETA”), which can be coated onto a carrier or surface such as a glass cover slip for example.
- DETA was coated on a glass surface to form a self-assembled monolayer.
- DETA has previously been shown to support neuronal (26), skeletal muscle (27), endothelial (38), and cardiac cell growth (39), and has been used in creating high-resolution, in vitro patterned circuits of embryonic hippocampus neurons (40).
- DETA substrates have been shown to promote guided axonal growth and direct axonal and dendritic process extension at the level of a single neuron (41). Therefore, the successful formation of NMJ on this substrate implies that some co-cultures of the invention can be patterned at high resolution to study engineered in vitro NMJs. Especially, this surface modification technique can be used for guiding specific NMJ formation.
- embodiments of the invention have many advantages. Some, but not all, of those advantages are listed here. Not all of these advantages are required by all embodiments of the invention.
- embodiments of the invention provide the first pure human based NMJ in vitro culture system. This human cell-based system bridges the gap between findings from animals and their clinical applications. The stem cell origin for both motoneurons and skeletal muscles enables the formation of these cultures in large quantities which can be important for high throughput drug screening. The serum-free medium allows this system to be highly reproducible and easy to manipulate. The patternable surface gives the power to the system to be engineered into neural circuits.
- Glass coverslips (6661 F52, 22x22 mm No. 1; Thomas Scientific, Swedesboro, NJ, USA) were cleaned using HCl/methanol (1:1) for at least 2 hours, rinsed with water, soaked in concentrated H2SO4 for at least 2 hours and rinsed with water. Coverslips were boiled in nanopure water and then oven dried.
- the trimethoxysilylpropyldiethylenetri-amine (DETA, T2910KG; United Chemical Technologies Inc., Bristol, PA, USA) film was formed by the reaction of cleaned surfaces with a 0.1% (v/v) mixture of the organosilane in freshly distilled toluene (T2904; Fisher, Suwanne, GA, USA).
- the DETA coated coverslips were heated to ⁇ 80oC, then cooled to room temperature (RT), rinsed with toluene, reheated to approximately the same temperature, and then cured for at least 2 hours at 110 C.
- Surfaces were characterized by contact angle and X-ray photoelectron 5 spectroscopy as described previously (26, 42, 43).
- the human spinal cord stem cell line was isolated and established as described in (44-46). MNs were differentiated from this cell line as described in (20). Briefly, ⁇ 1x106 hSCSCs were plated in one 60 mm paranox cell culture dish (Nunc, Cat #174888) and differentiated 4 days in the priming media followed by 6 days in differentiation media. The composition of the priming media and differentiation media were described in (20).
- hSKM SCs Human skeletal muscle stem cells
- Myoblast fusion into postmitotic myofibers was induced by incubation in differentiation medium (high-glucose DMEM (Invitrogen, Carlsbad, CA) supplemented with insulin (10 ⁇ g/ml), bovine serum albumin (50 ⁇ g/ml), epidermal growth factor (10 ng/ml) and gentamicin (50 ⁇ g/ml)).
- differentiation medium high-glucose DMEM (Invitrogen, Carlsbad, CA) supplemented with insulin (10 ⁇ g/ml), bovine serum albumin (50 ⁇ g/ml), epidermal growth factor (10 ng/ml) and gentamicin (50 ⁇ g/ml)
- insulin 10 ⁇ g/ml
- bovine serum albumin 50 ⁇ g/ml
- epidermal growth factor 10 ng/ml
- gentamicin 50 ⁇ g/ml
- differentiated hSCs were harvested and plated on top of these induced myotubes in a density of 200 cells/mm2, and the medium was changed to co-culture medium (Table 1). Two days later, the medium was fed by co-culture medium (without G5) by changing half of the medium. After another two days and thereafter, the cultures were fed by NBactive4 (Brain Bits) by changing half of the medium.
- NBactive4 Brain Bits
- motoneurons and skeletal muscles were described in detail in the Material and Methods. Briefly, human SKM stem cells were allowed to grow to confluence before induction of differentiation ( FIG. 1A ). After switching to differentiation media, the fusion of myocytes was initiated. Multi-nuclei myotubes formed gradually and were prevalent from day 4 in the culture ( FIG. 1B ). Differentiated human motoneurons (hMNs) were cultured as in Guo et al. (20) and were plated on the top of the differentiated myotubes and the medium was switched to a co-culture medium at this time. Both hSKMs and hMNs survived well in the co-culture media ( FIG. 1C ).
- Nonspecific binding sites were blocked in Blocking Buffer (5% Donkey serum plus 0.5% BSA in PBS) for 45 min at room temperature. Cells were then incubated with primary antibodies overnight at 4oC. After being washed with PBS 3x10 min, the cells were incubated with secondary antibodies for 2.5 hours at room temperature. The cells were then washed with PBS 3x10 min and mounted with Vectorshield with 4'-6-Diamidino-2-Phenylindole (dapi) (Vector laboratories, Inc.). Primary antibodies used in this study include: Rabbit-anti- ⁇ III Tubulin (Sigma, 1:1500), Mouse-anti-synaptophysin (Antibodies Inc., 1:100).
- the monoclonal antibody against muscle heavy chain (MHC, F1.625, 1:10) was obtained from the Developmental Studies Hybridoma Bank which is under the auspices of the NICHD and maintained by the University of Iowa.
- Secondary antibodies include: Donkey-anti-Mouse-488 (Invitrogen, 1:250) and Donkey-anti-Rabbit-594 (Invitrogen, 1:250). All antibodies were diluted in Blocking Buffer.
- Electrophysiological properties of spinal cord stem cell-derived motoneurons and human myotubes were investigated after ⁇ 10 days in the coculture using whole-cell patch-clamp recording techniques (26). The recordings were performed in a recording chamber located on the stage of a Zeiss Axioscope 2FS Plus upright microscope (50).
- Motoneurons were identified visually under an infrared DICvideomicroscope. The largest multipolar or round cells (15-25 ⁇ m diam) with bright illuminance in the culture were tentatively identified as motoneurons (51, 52). Patch pipettes with a resistance of 6-10 MO were made from borosilicate glass (BF 150-86-10; Sutter, Novato, CA) with a Sutter P97 pipette puller (Sutter Instrument Company).
- the electrophysiological properties of the MNs and myotubes in the co-culture were evaluated using voltage and current clamp recordings for each cellular component. Representative voltage-clamp and current-clamp recordings for the MNs and myotubes are shown in FIG. 4 .
- the electrical properties of MNs in the co-culture system such as membrane resistance, resting membrane potential, Na+/K+ current amplitude, the ability to repetitively fire and the amplitude of action potential (AP), were comparable to results described previously (20, 26).
- the electrical properties for the myotubes were also comparable to previously published results (27).
- Video 1 (10 min), during the first 6 min, the myotube contracted in pulses which was recurrent approximately every 1-2 min. The contraction then stopped after this time period.
- Video 2 (15 min), the contraction pulses of three spots were observed to intermittently contract.
- Video 3 (15 min), myotube contraction was recorded for 7 min. The addition of Curare (5 ⁇ M) silenced the contraction.
- Video 4 22 min
- any temperature, weight, volume, time interval, pH, salinity, molarity or molality, range, concentration and any other measurements, quantities or numerical figures expressed herein are intended to be approximate and not an exact or critical figure unless expressly stated to the contrary.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- Neurology (AREA)
- Biophysics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Neurosurgery (AREA)
- Veterinary Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Physical Education & Sports Medicine (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
Claims (14)
- Procédé de formation de jonctions neuromusculaires, le procédé comprenant :la formation de jonctions neuromusculaires fonctionnelles entre des motoneurones et des cellules musculaires par co-culture d'un ou de plusieurs motoneurones humains dérivés de cellules souches de moelle épinière humaine et d'une ou de plusieurs cellules musculaires humaines dérivées de cellules souches de muscle squelettique humain dans un milieu sensiblement exempt de sérum, dans lequel le milieu comprend au moins un constituant favorisant la synaptogenèse et un ou plusieurs facteurs trophiques.
- Procédé de formation de jonctions neuromusculaires, le procédé comprenant :la mise en suspension de cellules de muscle squelettique humain dérivées de cellules souches de muscle squelettique humain dans un milieu sans sérum ;la mise en suspension de motoneurones humains dérivés de cellules souches de moelle épinière humaine dans le milieu sans sérum ;l'étalement des cellules musculaires en suspension et des motoneurones en suspension sur un support artificiel ; etla surveillance de la formation de jonctions neuromusculaires fonctionnelles.
- Procédé selon la revendication 1, dans lequel le milieu ne contient pas de sérum.
- Procédé selon l'une quelconque des revendications 1 à 3, comprenant en outre l'ajout de NbActiv4 au milieu.
- Procédé selon la revendication 2 ou 4, dans lequel le support artificiel comprend une monocouche de substrat à base de silicium déposée sur celui-ci.
- Procédé selon la revendication 5, dans lequel la monocouche de substrat à base de silicium comprend de la DETA.
- Procédé selon la revendication 6, dans lequel la monocouche de substrat à base de silicium est déposée sur le support artificiel selon un motif prédéterminé.
- Procédé selon l'une quelconque des revendications précédentes, dans lequel le milieu comprend au moins un constituant choisi parmi le GDNF, le BDNF, le Shh, RA, l'IGF-1, l'AMPc, le CNTF, la NT-3, la NT-4, la Vitronectine, la laminine, le G5 et l'Agrine.
- Système de co-culture pour former une jonction neuromusculaire de mammifère synthétique comprenant : un motoneurone humain dérivé d'une cellule souche de moelle épinière humaine et une cellule de muscle squelettique humain provenant d'une cellule souche de muscle squelettique humain, dans un milieu sensiblement exempt de sérum.
- Système de co-culture selon la revendication 9, dans lequel le motoneurone humain est lié de manière fonctionnelle à la cellule musculaire humaine sur une surface artificielle.
- Système de co-culture selon la revendication 10, dans lequel la surface artificielle a un substrat monocouche à base de silicium déposé sur celle-ci.
- Système de co-culture selon la revendication 11, dans lequel le substrat monocouche à base de silicium est déposé selon un motif prédéterminé.
- Système de co-culture selon la revendication 11 ou la revendication 12, dans lequel le substrat monocouche à base de silicium comprend de la DETA.
- Système de co-culture selon la revendication 9, dans lequel le milieu sans sérum comprend en outre du NbActiv4.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12/765,996 US8815584B1 (en) | 2009-04-23 | 2010-04-23 | Method of co-culturing mammalian muscle cells and motoneurons |
US33200310P | 2010-05-06 | 2010-05-06 | |
PCT/US2011/035585 WO2011133985A1 (fr) | 2010-04-23 | 2011-05-06 | Formation de jonctions neuromusculaires dans un système défini |
Publications (3)
Publication Number | Publication Date |
---|---|
EP2585171A1 EP2585171A1 (fr) | 2013-05-01 |
EP2585171A4 EP2585171A4 (fr) | 2013-11-20 |
EP2585171B1 true EP2585171B1 (fr) | 2018-12-26 |
Family
ID=44761205
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP11772857.6A Active EP2585171B1 (fr) | 2010-04-23 | 2011-05-06 | Formation de jonctions neuromusculaires dans un système défini |
Country Status (4)
Country | Link |
---|---|
US (3) | US9952204B2 (fr) |
EP (1) | EP2585171B1 (fr) |
CA (1) | CA2798777C (fr) |
WO (1) | WO2011133985A1 (fr) |
Families Citing this family (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10386360B2 (en) | 2009-03-13 | 2019-08-20 | University Of Central Florida Research Foundation, Inc. | Bio-microelectromechanical system transducer and associated methods |
US9163216B1 (en) | 2009-04-23 | 2015-10-20 | University Of Central Florida Research Foundation, Inc. | Method for culturing skeletal muscle for tissue engineering |
US8815584B1 (en) | 2009-04-23 | 2014-08-26 | University Of Central Florida Research Foundation, Inc. | Method of co-culturing mammalian muscle cells and motoneurons |
US8828721B1 (en) | 2009-05-28 | 2014-09-09 | University Of Central Florida Research Foundation, Inc. | Method of myelinating isolated motoneurons |
US9404140B1 (en) | 2009-11-03 | 2016-08-02 | The University Of Central Florida Research Foundation, Inc. | Patterned cardiomyocyte culture on microelectrode array |
CA2788905A1 (fr) | 2010-02-05 | 2011-08-11 | University Of Central Florida Research Foundation, Inc. | Modele et procedes pour l'identification de points d'action dans des cellules electriquement actives |
CA2798777C (fr) | 2010-04-23 | 2016-09-06 | University Of Central Florida Research Foundation, Inc. | Formation de jonctions neuromusculaires dans un systeme defini |
US9861663B2 (en) * | 2012-02-23 | 2018-01-09 | Technion Research & Development Foundation Ltd. | Ex-vivo vascularized implant composition comprising poly-l-lactic acid, polylactic-co-glycolic-acid and olfactory bulb cells |
EP2951281B1 (fr) | 2013-01-30 | 2018-06-27 | University of Central Florida Research Foundation, Inc. | Système et procédé pour imiter une fonction cardiaque |
US10935541B2 (en) | 2014-08-07 | 2021-03-02 | University Of Central Florida Research Foundation, Inc. | Devices and methods comprising neuromuscular junctions |
US10542961B2 (en) | 2015-06-15 | 2020-01-28 | The Research Foundation For The State University Of New York | System and method for infrasonic cardiac monitoring |
WO2018038987A1 (fr) | 2016-08-26 | 2018-03-01 | University Of Central Florida Research Foundation, Inc. | Système in vitro à composants multiples pour déduire des voies de signalisation cellulaires par des motifs de stimulation électronique |
EP3562591A4 (fr) | 2016-12-30 | 2020-08-19 | University of Central Florida Research Foundation, Inc. | Système d'organe sur puce microfluidique sans pompe comprenant un système immunitaire fonctionnel |
US10767164B2 (en) | 2017-03-30 | 2020-09-08 | The Research Foundation For The State University Of New York | Microenvironments for self-assembly of islet organoids from stem cells differentiation |
WO2019078263A1 (fr) * | 2017-10-17 | 2019-04-25 | 国立大学法人京都大学 | Procédé d'obtention de jonction neuromusculaire artificielle à partir de cellules souches pluripotentes |
CN112877282A (zh) * | 2021-02-09 | 2021-06-01 | 南通大学 | 一种体外培养原代神经肌肉接头的方法 |
Family Cites Families (44)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5682899A (en) | 1991-05-16 | 1997-11-04 | Ami-Med Corporation | Apparatus and method for continuous cardiac output monitoring |
US5442510A (en) | 1993-06-23 | 1995-08-15 | The United States Of America As Represented By The Secretary Of The Navy | Control system for tracking nonlinear systems |
US20090239940A1 (en) | 1997-07-22 | 2009-09-24 | Del Monte Federica | Treating heart failure and ventricular arrhythmias |
US7923015B2 (en) | 1997-08-14 | 2011-04-12 | Institut Pasteur | Methods for direct visualization of active synapses |
US5948621A (en) | 1997-09-30 | 1999-09-07 | The United States Of America As Represented By The Secretary Of The Navy | Direct molecular patterning using a micro-stamp gel |
US7266457B1 (en) | 1999-05-21 | 2007-09-04 | Hesperos, Llc | High throughput functional genomics |
WO2001029206A1 (fr) | 1999-10-15 | 2001-04-26 | Advanced Cell Technology, Inc. | Procedes de production de cellules souches et de cellules embryonnaires a lignee defectueuse differenciees |
EP1240525A2 (fr) | 1999-12-23 | 2002-09-18 | PHARMACIA & UPJOHN COMPANY | Titrages et methodes diagnostiques et therapeutiques bases sur l'utilisation des canaux sodiques comme cibles de proteine beta-amyloide ou d'agregats de celle-ci |
CA2450376A1 (fr) | 2001-04-20 | 2002-10-31 | The Board Of Regents Of The University Of Oklahoma | Neuromodulation cardiaque et procedes d'utilisation correspondants |
US6844028B2 (en) | 2001-06-26 | 2005-01-18 | Accelr8 Technology Corporation | Functional surface coating |
CA2457323A1 (fr) | 2001-08-06 | 2003-08-21 | Vanderbilt University | Systemes et procedes de discrimination d'un agent |
US6866383B2 (en) | 2001-08-17 | 2005-03-15 | Avery Dennison Corporation | Topcoat compositions, substrates containing a topcoat derived therefrom, and methods of preparing the same |
US6916541B2 (en) | 2001-09-07 | 2005-07-12 | Penn State Research Foundation | Modified substrates for the attachment of biomolecules |
US8153424B2 (en) | 2001-10-03 | 2012-04-10 | Wisconsin Alumni Research Foundation | Method of in vitro differentiation of neural stem cells, motor neurons and dopamine neurons from primate embryonic stem cells |
US20030144823A1 (en) | 2001-11-01 | 2003-07-31 | Fox Jeffrey J. | Scale-free network inference methods |
WO2003081204A2 (fr) | 2002-03-20 | 2003-10-02 | Purdue Research Foundation | Microdetecteur et dispositif s'y rapportant, et procede de fabrication |
US20040076961A1 (en) | 2002-10-21 | 2004-04-22 | Lewis Mark A. | Biomolecule retaining material and methods for attaching biomolecules to a surface |
US7341841B2 (en) | 2003-07-12 | 2008-03-11 | Accelr8 Technology Corporation | Rapid microbial detection and antimicrobial susceptibility testing |
WO2005010172A2 (fr) | 2003-07-16 | 2005-02-03 | Boston Scientific Limited | Structures d'echafaudage alignees pour regeneration myocardique accrue |
US7198855B2 (en) | 2003-09-12 | 2007-04-03 | Becton, Dickinson And Company | Methods of surface modification of a flexible substrate to enhance cell adhesion |
EP3070174B1 (fr) | 2004-05-11 | 2019-11-06 | Ncardia AG | Découverte de médicaments utilisant des cellules différentiées in vitro |
US7807462B2 (en) | 2004-11-16 | 2010-10-05 | University Of Medicine And Dentistry Of New Jersey | Method for producing a functional neuron |
US7691629B2 (en) | 2004-11-17 | 2010-04-06 | Neuralstem, Inc. | Transplantation of human neural cells for treatment of neurodegenerative conditions |
US7718846B2 (en) | 2005-05-16 | 2010-05-18 | Gideon Koren | Animal models of long QT syndrome and uses thereof |
JP2009509124A (ja) | 2005-06-16 | 2009-03-05 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | 大型のパラレルの免疫ベースのアレルギー試験および蛍光のエバネセント場励起のためのデバイス |
NZ565683A (en) | 2005-07-08 | 2011-04-29 | Braincells Inc | Methods for identifying agents and conditions that modulate neurogenesis in human neural cells |
EP1941031B1 (fr) | 2005-10-05 | 2013-12-25 | The Board Of Trustees Of The University Of Illinois | Cellules souches de type embryonnaire isolées dérivées du sang du cordon ombilical humain |
US20070129447A1 (en) | 2005-12-02 | 2007-06-07 | Sra Jasbir S | Method of drug therapy in treatment of cardiac arrhythmias |
AU2006326853B2 (en) | 2005-12-22 | 2012-02-16 | Es Cell International Pte Ltd | Direct differentiation of cardiomyocytes from human embryonic stem cells |
WO2007098065A2 (fr) | 2006-02-17 | 2007-08-30 | The United States Of America As Represented By The Department Of Veterans Affairs | Isoformes de canaux sodium humains |
US7927671B2 (en) | 2006-09-21 | 2011-04-19 | Chisso Corporation | Trifunctional compound, composition and polymer thereof |
US8193225B2 (en) | 2006-10-13 | 2012-06-05 | The Board Of Regents Of The University Of Texas System | Isoxazole amides, derivatives and methods of chemical induction of neurogenesis |
EP2139990A1 (fr) * | 2007-03-23 | 2010-01-06 | California Stem Cell, Inc. | Cellules humaines de progéniteur de neurone moteur de stade avancé et procédés de préparation et d'utilisation de celles-ci |
CN101835479A (zh) | 2007-07-25 | 2010-09-15 | 佰欧益有限公司 | 多系祖细胞分化为软骨细胞 |
WO2009114133A1 (fr) | 2008-03-10 | 2009-09-17 | The J. David Gladstone Institutes | Cellules et essais pour une utilisation dans la détection du syndrome du qt long |
ES2442168T3 (es) * | 2008-12-05 | 2014-02-10 | Yeda Research And Development Co. Ltd. | Métodos de diagnóstico de enfermedades de neuronas motoras |
WO2010127280A1 (fr) | 2009-05-01 | 2010-11-04 | President And Fellows Of Harvard College | Dosages à haut débit pour déterminer la fonction contractile et dispositifs pour les utiliser |
EP2434896A4 (fr) | 2009-05-28 | 2014-01-22 | Univ Central Florida Res Found | Production in vitro d'oligodendrocytes à partir de cellules souches de cordon ombilical humain |
WO2010138679A1 (fr) | 2009-05-28 | 2010-12-02 | University Of Central Florida Research Foundation, Inc. | Procédé de criblage de médicaments pour une inversion de la neurotoxicité de l'amyloïde bêta |
CA2788905A1 (fr) | 2010-02-05 | 2011-08-11 | University Of Central Florida Research Foundation, Inc. | Modele et procedes pour l'identification de points d'action dans des cellules electriquement actives |
CA2798777C (fr) | 2010-04-23 | 2016-09-06 | University Of Central Florida Research Foundation, Inc. | Formation de jonctions neuromusculaires dans un systeme defini |
WO2012158923A1 (fr) | 2011-05-17 | 2012-11-22 | University Of Central Florida Research Foundation, Inc. | Neurones électriquement actifs stables provenant de tissus adultes |
CN103987854A (zh) | 2011-07-21 | 2014-08-13 | 小利兰·斯坦福大学托管委员会 | 来自患者的诱导性多能干细胞的心肌细胞及其使用方法 |
EP2885394A4 (fr) | 2012-08-17 | 2016-04-20 | Univ Central Florida Res Found | Procédés, systèmes et compositions pour des modèles cellulaires in vitro fonctionnels de systèmes de mammifère |
-
2011
- 2011-05-06 CA CA2798777A patent/CA2798777C/fr active Active
- 2011-05-06 WO PCT/US2011/035585 patent/WO2011133985A1/fr active Application Filing
- 2011-05-06 US US13/102,672 patent/US9952204B2/en active Active
- 2011-05-06 US US13/696,396 patent/US8835168B2/en active Active
- 2011-05-06 EP EP11772857.6A patent/EP2585171B1/fr active Active
-
2014
- 2014-08-06 US US14/453,207 patent/US9267936B2/en active Active
Non-Patent Citations (1)
Title |
---|
None * |
Also Published As
Publication number | Publication date |
---|---|
US20130115694A1 (en) | 2013-05-09 |
US9267936B2 (en) | 2016-02-23 |
WO2011133985A1 (fr) | 2011-10-27 |
US20140349884A1 (en) | 2014-11-27 |
CA2798777A1 (fr) | 2011-10-27 |
US8835168B2 (en) | 2014-09-16 |
US9952204B2 (en) | 2018-04-24 |
EP2585171A1 (fr) | 2013-05-01 |
EP2585171A4 (fr) | 2013-11-20 |
US20110250682A1 (en) | 2011-10-13 |
CA2798777C (fr) | 2016-09-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP2585171B1 (fr) | Formation de jonctions neuromusculaires dans un système défini | |
Guo et al. | Neuromuscular junction formation between human stem cell-derived motoneurons and human skeletal muscle in a defined system | |
Guo et al. | Neuromuscular junction formation between human stem-cell-derived motoneurons and rat skeletal muscle in a defined system | |
Steinbeck et al. | Functional connectivity under optogenetic control allows modeling of human neuromuscular disease | |
Heikkilä et al. | Human embryonic stem cell-derived neuronal cells form spontaneously active neuronal networks in vitro | |
Lindsay | Nerve growth factors (NGF, BDNF) enhance axonal regeneration but are not required for survival of adult sensory neurons | |
Southam et al. | Microfluidic primary culture model of the lower motor neuron–neuromuscular junction circuit | |
Kleitman et al. | Schwann cell surfaces but not extracellular matrix organized by Schwann cells support neurite outgrowth from embryonic rat retina | |
Das et al. | A defined system to allow skeletal muscle differentiation and subsequent integration with silicon microstructures | |
KR101204188B1 (ko) | 네스틴 발현 모낭 줄기 세포 | |
Cooke et al. | Neural differentiation regulated by biomimetic surfaces presenting motifs of extracellular matrix proteins | |
US10266804B2 (en) | Method of co-culturing mammalian muscle cells and motoneurons | |
Luo et al. | Electrospun nanofibers facilitate better alignment, differentiation, and long-term culture in an in vitro model of the neuromuscular junction (NMJ) | |
Das et al. | Skeletal muscle tissue engineering: a maturation model promoting long-term survival of myotubes, structural development of the excitation–contraction coupling apparatus and neonatal myosin heavy chain expression | |
Guo et al. | Tissue engineering the monosynaptic circuit of the stretch reflex arc with co-culture of embryonic motoneurons and proprioceptive sensory neurons | |
Saito-Diaz et al. | Derivation of peripheral nociceptive, mechanoreceptive, and proprioceptive sensory neurons from the same culture of human pluripotent stem cells | |
US10160953B2 (en) | Method for culturing skeletal muscle for tissue engineering | |
EP3415618B1 (fr) | Procédés de production d'organoïdes neuronale par génie biologique (benos) et leurs utilisations | |
Bucchia et al. | Limitations and challenges in modeling diseases involving spinal motor neuron degeneration in vitro | |
Guo et al. | Tissue engineering the mechanosensory circuit of the stretch reflex arc with human stem cells: Sensory neuron innervation of intrafusal muscle fibers | |
Guy et al. | Human neural organoids: Models for developmental neurobiology and disease | |
CA3001242A1 (fr) | Procedes in vitro d'identification de modulateurs de l'activite de jonction neuromusculaire | |
Schutte et al. | Astrocyte-enriched feeder layers from cryopreserved cells support differentiation of spontaneously active networks of human iPSC-derived neurons | |
Poulin et al. | Differentiation of lymphoblastoid-derived iPSCs into functional cardiomyocytes, neurons and myoblasts | |
Rumsey et al. | Tissue engineering intrafusal fibers: Dose-and time-dependent differentiation of nuclear bag fibers in a defined in vitro system using neuregulin 1-β-1 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20121119 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
DAX | Request for extension of the european patent (deleted) | ||
RIC1 | Information provided on ipc code assigned before grant |
Ipc: C12N 5/079 20100101ALI20131009BHEP Ipc: A61P 21/00 20060101AFI20131009BHEP Ipc: C12N 5/077 20100101ALI20131009BHEP |
|
A4 | Supplementary search report drawn up and despatched |
Effective date: 20131018 |
|
17Q | First examination report despatched |
Effective date: 20160223 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: EXAMINATION IS IN PROGRESS |
|
GRAP | Despatch of communication of intention to grant a patent |
Free format text: ORIGINAL CODE: EPIDOSNIGR1 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: GRANT OF PATENT IS INTENDED |
|
INTG | Intention to grant announced |
Effective date: 20180719 |
|
GRAS | Grant fee paid |
Free format text: ORIGINAL CODE: EPIDOSNIGR3 |
|
GRAA | (expected) grant |
Free format text: ORIGINAL CODE: 0009210 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE PATENT HAS BEEN GRANTED |
|
AK | Designated contracting states |
Kind code of ref document: B1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
REG | Reference to a national code |
Ref country code: GB Ref legal event code: FG4D |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: EP |
|
REG | Reference to a national code |
Ref country code: AT Ref legal event code: REF Ref document number: 1080588 Country of ref document: AT Kind code of ref document: T Effective date: 20190115 |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R096 Ref document number: 602011055172 Country of ref document: DE |
|
REG | Reference to a national code |
Ref country code: IE Ref legal event code: FG4D |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: NV Representative=s name: VALIPAT S.A. GEVERS SA, CH |
|
REG | Reference to a national code |
Ref country code: NL Ref legal event code: FP |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: PCAR Free format text: NEW ADDRESS: RUE DES NOYERS 11, 2000 NEUCHATEL (CH) |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: FI Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 Ref country code: BG Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20190326 Ref country code: LT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 Ref country code: LV Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 Ref country code: HR Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 Ref country code: NO Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20190326 |
|
REG | Reference to a national code |
Ref country code: LT Ref legal event code: MG4D |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: GR Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20190327 Ref country code: RS Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 Ref country code: SE Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 Ref country code: AL Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 |
|
REG | Reference to a national code |
Ref country code: AT Ref legal event code: MK05 Ref document number: 1080588 Country of ref document: AT Kind code of ref document: T Effective date: 20181226 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: CZ Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 Ref country code: PT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20190426 Ref country code: IT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 Ref country code: ES Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 Ref country code: PL Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: SK Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 Ref country code: RO Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 Ref country code: IS Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20190426 Ref country code: EE Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 Ref country code: SM Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R097 Ref document number: 602011055172 Country of ref document: DE |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: DK Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 Ref country code: AT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 |
|
PLBE | No opposition filed within time limit |
Free format text: ORIGINAL CODE: 0009261 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT |
|
26N | No opposition filed |
Effective date: 20190927 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: MC Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: LU Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20190506 Ref country code: SI Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: TR Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: IE Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20190506 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: CY Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: MT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 Ref country code: HU Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO Effective date: 20110506 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: MK Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20181226 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: FR Payment date: 20230309 Year of fee payment: 13 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: GB Payment date: 20230316 Year of fee payment: 13 |
|
P01 | Opt-out of the competence of the unified patent court (upc) registered |
Effective date: 20230523 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: NL Payment date: 20230314 Year of fee payment: 13 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: DE Payment date: 20230314 Year of fee payment: 13 Ref country code: CH Payment date: 20230602 Year of fee payment: 13 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: BE Payment date: 20230418 Year of fee payment: 13 |